Mitochondria are organelles present in eukaryotic cells and they play a role in synthesizing ATP, which is energy source between generated by oxidative phosphorylation of substances, through an electron transport system.
Mitochondria contain high levels of reactive oxygen species as by-products of oxygen respiration. Reactive oxygen species are continually damaging mitochondrial DNA oxidatively. In order to maintain their DNA and their functions as organelles, mitochondria must have a mechanism for repairing such damage to DNA and suppressing mitochondrial DNA abnormalities. Homologous recombination is believed to be indispensable in the repairing of eukaryotic nuclear DNA as well as bacterial and viral DNA.
Homologous recombination is a phenomenon universally observed in the world of organisms. Homologous recombination may occur, when a DNA molecule has a region having an almost identical base sequence with a base sequence of a region of another DNA molecule allowing the, pairing or the DNA molecules over several hundred base pairs. DNA chains of the two molecules are cut and re-linked to a different chain from the initial one at a site correctly corresponding with each other in those regions. As a result of homologous recombination, damage to one DNA molecule may be complemented by the other DNA. Thus, it is very likely that homologous recombination could play an important and general role in mitochondrial DNA repair.
When the activity of a gene inducing homologous recombination (hereinafter referred to as a "homologous recombination gene") is reduced, it becomes impossible to repair damaged DNA. Thus DNA abnormalities will increase. Furthermore, when a homologous recombination gene functions abnormally, abnormal unequal crossover will occur between a pair of repeated sequences located at sites other than those of alleles. As a result, DNA abnormalities such as deletions are induced. Deletion in mitochondrial DNA has been shown to be associated with mitochondrial diseases (e.g., mitochondrial myopathy which is caused by lack of an enzyme in the energy producing system of mitochondria), aging and the like Holt, I. J. et al., Nature (London), 331:717-719 (1988)!.
Therefore, by isolating a mutant gene involved in those abnormalities in homologous recombination described above and finding out the role of such a gene, it will become possible to use the detection and modification of such a mutant gene in the diagnosis or treatment of mitchondria-related diseases as well as in the prevention of aging and the like.
Most of mitochondrial proteins are encoded by DNAs located on nuclear chromosomes. It is thus expected that the homologous recombination in mitochondrial will be dependent on the genetic information contained in nuclei.
Therefore, as one step to elucidate the role and mechanism of mitochondrial homologous recombination, it is necessary to isolate a gene having a mutation which causes a defect in mitochondrial homologous recombination, i.e., a mutant gene in nuclei.
Conventionally, yeast has been considered as the most appropriate system for obtaining genetic information concerning mitochondria (information on the role of mitochondrial homologous recombination).
Even in yeast, however, it has been extremely difficult to examine a large number of clones for a mutant gene, in particular a recessive mutant gene in a nuclear chromosome involved in mitochondrial homologous recombination. In order to identify such a gene, it is necessary to carry out a mating experiment for detecting the recombination of mitochondria. However, when cells having a recessive mutant gene for mitochondrial homologous recombination are mated with cells having a normal gene for the recombination, the recessive mutant gene is hidden by the function of the normal gene and thus the recessive mutant gene cannot be detected.
Therefore, in the conventional method for isolating a recessive mutant gene in a nuclear chromosome, the following operations are required for the isolation; i) constructing monoploid cells of both alleic mating types (a and .alpha.) each having the same nuclear genotype as that of respective mutant monoploid cells; ii) introducing mitochondria having different gene markers into either an a- or .alpha.-derivative through the isolation and cell fusion experiments of a .rho.-derivative (derivative having no mitochondrial gene) ; and iii) carrying out a mating experiment between a-monoploid and .alpha.-monoploid in order to determine the recombination frequency of the mitochondrial gene markers. However, these operations are complicated and time-consuming and it has been extremely difficult to examine a large number of candidate cells simultaneously.